Assaying apparatus



Jan. 4, 1966 A. B. sALlsBURY, JR., ETAL 3,227,522

SSAYING APPARATUS Filed Sept. 19, 1961 2 Sheets-Sheet 1 @www Jan. 4,1966 A, B. sALlsBuRY, JR., ETAL 3,227,522

ASSAYING APPARATUS Filed Sept. 19. 1961 2 Sheets-Sheet 2 fr-cey UnitedStates Patent O 3,227,522 ASSAYNG APPARAUS Alvin B. Salisbury, Jr.,Fairborn, Guy A. Baiser, Dayton,

and eClifford I. Clt, Xenia, Ohio, assiguors to Laboratories, line.,Fairborn, Ohio Filed Sept. 19, 1951, Ser. No, 139,211? 6 Ciaims. (Ci.23-253) This invention relates generally to apparatus for assaying thereactions of different agents, for example, chemical agents, to eachother and, more particularly, to apparatus for so-called agar diffusionassays in which a fiat surface of an agar is impregnated with one agentand the effect of one or more testing agents on the first agent isobtained by bringing the other agents into contact with spaced portionsof the surface so that they diffuse into the agar and produce a visiblereaction with the tirst agent. Such reactions may be visiele as changesof color or as the presence or absence or" growth where microorganismsconstitute the rst agent and the other agents are growth inhibiting orencouraging substances.

A general object of the invention is to provide novel apparatus of theabove character which, compared to similar prior apparatus, insures amore uniform and controlled distribution of each testing agent on theagar surface and thereby achieves more accurate and reliable results.

Another object is to support each testing agent on a projecting postwhich is constructed in a novel manner to insure Contact of a controlledquantity of the agent with the agar surface and to avoid reduction ofthat quantity or shifting of the agent on the agar due to air bubblestrapped on the surface.

A further detailed object is to proportion the parts of the projectionin a novel manner to obtain the desired conv-ex contour while avoidingcracks in the agar.

The Vinvention also resides in the novel and simple manner of formingand connecting a plurality of projections for use with standardcommercially available assay apparatus of the Petri dish type includingan enclosing lid and inverted during incubation to avoid moisturecollecting on the lid and dropping onto the agar.

Other objects and advantages of the invention will become apparent fromthe following detailed description taken in connection with theaccompanying drawings in which FiG. l is a plan view of the test agentssupporting plate embodying the novel features of the present invention.

FIG. 2 is a side elevation of the plate.

FlG. 3 is a fragmentary sectional view taken along the line 3 3 of FIG.1 and showing an agent supporting projection.

FG. 4 is a perspective view of the plate and position on agar in a Petridish bottom.

FlG. 5 is a plan view oft -e parts in FEG. 4.

FlG. 6 is a sectional view of plate in contact with agar in a completePetri dish inverted for incubation.

FIG. 7 is a view similar to FIG. 5 with the plate removed.

FIGS. 8 and 9 are fragmentary sectional views taken along line 8 8 fFiG. 5 and showing successive positions of a projection with respect tothe agar after different lengths of time of contact between the two.

FIGS. 10 and 11 are views `similar to FGS. 8 and 9 and showing -amodified proiection configurations.

FG. l2 is a side elevational view of a stack of plates some parts ofwhich have been `broken away and shown in section.

FIGS. 13 and 14 are plan views of modified plates of different size withdifferent number of agents supporting projections.

The present invention is especially suited for agar diffusion assays inwhich an agar medium 16 having a at exposed surface 17 is impregnatedwith an agent to be ested and quantities of one or more other agents areplaced on the surface in spaced relation for observation of the effectof the other agents on the first agent. Such assays may be conducted forvarious purposes, one of the more common being biological assays todetermine the effects on microorganisms of growth inhibiting orencouraging agents. Another is spot test analysis of solutionscontaining diiferent trace metals, complexes or ions. These are placedon the agar and suitable indica-tors forming the other test agents areplaced at spaced points on the agar to produce chemical reactions suchas visible color changes.

ln the case of microbiological assays, the agar usually is contained ina Petri dish having a bottom 18 with upstanding sides. The agar, as iswell known, is a gelatinous material partially filling the bottom of thedish and containing moisture. The exposed agar surface 1'7 is streakedwith bacterial inoculum to be tested and a quantity of one or mor-egrowth affecting agents is placed on the surface at a point spaced fromother agents. The dish bottom then is covered with a lid 19 and theassembly is incubated, the agents diffusing into the agar anddiscouraging or encouraging the growth of the bacteria depending on thetype of agent used. Examples of inhibiting agents are antibiotics,sulfonamides and other chemotherapeutic substances. Some growthencouraging agents are carbohydrates, proteins, fats, vitamins,horins-nes, etc.

The visual effects of inhibitors on the growth of bacteria isillustrated in FIGS. 5, 6 and 7. Referring to these figures, differentinhibitors carried by supports 26 are brought into contact with spacedportions of the agar surface 17 and diffuse into tbc agar as indicatedby the dotted spaces 21. These spaces represent zones where the growthof the bacteria has been inhibited, the sizes of the zones varying withthe effectiveness of the different agents. The absence of zones ofinhibition around two of the supports indicates that the agents on thesesupports had no inhibiting effect on the bacteria. ri`he reason 'forinverting the bottom 18 and cover 19 of the Petri dish as shown in FIG.6 is to prevent moisture accumulation on the inside cover surface aswell as evaporation and loss of water from the agar during incubation.Handling of a plurality of agents is facilitated bythe provision of abody ember 22 which connects the supports and maintains them in adesired spaced relation. I

To obtain accurate and reliable test results, the present inventionVcontemplates a novel construction of each agent support 2i) insuringuniform and .complete diffusion of the agent into the agar 16 andavoiding shifting of the support with respect to the agar. The supportthus is formed as a projection which terminates in a continuous smoothsurface 23 of convex exterior contour covered by a test agent in theform of a thin layer. As the support is moved toward the exposed agarsurface 17, this configuration results in contact of the agent and theagar first at a central portion of the convex surface and thenprogressively to the peripheral portions, any air bubbles thus areadvanced ahead of the progressing contact line and do not becomeentrapped between the support and the agar so as to prevent contact ofthe agar surface by all of the agent or to space the end surface of theprotection from the agar surface. The projection thus is able to sinkpartially into the agar as shown in FEGS. 9 and 11 and prevent sidewiseshifting relative to the agar.

The preferred test agent supporting surface 23 as shown in FIGS. 1 to 9and 12 to 14 is of a rounded or spherical convex contour with a circularperiphery 24 (FIG. 3).

Its height, that is, the lateral or transverse spacing from theoutermost center portion to its periphery, is correlated with itsdiameter to provide the desired convexity to insure contact of all ofthe surface with the agar while avoiding a sharp point liable topenetrate the agar sufficiently to produce cracks resulting in errors inthe assay. A suitable height, indicated at A in FIG. 3, found to besatisfactory for a surface of 6.6 millimeters to 13 millimeters diameteris .001 to .010 of an inch or .0254 to .1524 of a millimeter.Convexities with heights outside of this range were found to be subjectto entrapment of air bubbles or cracking of the agar, heights of .003 to.006 of an inch being the most satisfactory for the prevention of airentrapment. A suitable length of each projection of this approxhnatesize is from two to five millimeters.

Another convex contour of agent supporting surface 23 found to besatisfactory is a conical surface as shown in a modification of FIGS.and 11. In this construction as in the preferred rounded surface, theheight or transverse spacing of the center of the surface and theperiphery is important to insure full contact of the agent with the agarand to avoid air bubbles without cracking the agar. With bothconfigurations, moisture collects between the center and periphery ofthe surface when the surface first contacts the exposed surface 17 ofthe agar. With the lapse of time, the agar surface beneath the convexsurface assumes a complementary concave contour resulting in a pit orrecess and tending further to reduce shifting of the projection relativeto the agar. Such formation of the recess beneath each projection isbelieved to be due to the weight of the projection and its connectingbody and also to what may be termed interfacial tension. As the pit isformed, the moisture collected between the projection and the agarassumes a more uniform thickness and forms a film 26 (FIGS. 9 and l1)which, due to capillary action or interfacial tension, resists movementof the projection away from the agar.

In another of its aspects, the present invention contemplates forming aplurality of the projecting supports 2li and their connecting body 22 ina novel manner enabling the capillary action or interfacial tension ofthe film 26 between the agent surface 23 and the exposed agar surface 17to retain the projections in position with respect to the agar when thecovered Petri dish is inverted for incubation as shown in FIG. 6. Theinvention further contemplates inexpensive manufacture of theprojections and body and simplification and accuracy in their use. Tothese ends, the body is formed as a thin sheet or plate and theprojections and supports are formed as integral parts therefo, theweight of the plate being less than the sum of the tension forces due tothe film 25 on the projections. Spaced portions of the sheet thus aredepressed laterally to form recesses on one side and the projections onthe other.

To facilitate insertion and withdrawal of the plate 22 between theupstanding walls of the Petri dish bottom 13, the plate has the sameperipheral shape as the dish but is smaller thereby leaving a spacebetween the two when the plate is inserted in the dish. This space isdistributed around the plate, that is, the plate is centered in thedish, by locating lugs 27 engageable with the dish wall and projectingradially and outwardly from angularly spaced ports of the periphery ofthe plate. If the plate is removed from the dish and is desired to bereinserted in its original position, the proper orientation is insuredby the provision of another projection 28 which is rectangular andtherefore provides a recess 29 on the agar (FIG. 7) easilydistinguishable from the circular recesses 25 of the agent carryingprojection 20.

A material found to be suitable for the plate 22 is a thermoplasticresin such as a polyvinyl chloride sheet .093 to .004 of an inch thick,which is known to be nonabsorbent. If desired, one side may be roughenedto provide a pleasing appearance while leaving the plate translucent topermit observation of the zone 21 of inhibi- CII tion through the plate.A plate of this material and thickness weighs less than the forceresulting from the capillary action or surface tension of the moisturebetween the agar lo and the projection 20 when inverted as shown in FIG.6. Shipping and storing of a plurality of plates is facilitated bymaking each projection between its end and the plate of tapered contour,the projections in this instance being conical with circular crosssections. The projections of one plate thus may nest in the hollowprojections of the next plate for stacking the plates in spaced relationand thereby keeping the agents on the projections of one plate out ofcontact with the projections of the next plate as shown in FIG. l2.

Various common commercial methods of forming the plate with itsprojections may be used, for example, stamping or vacuum forming usingmale or female dies with a sheet of the plastic material heated duringthe shaping and then cooled in a well known manner.

The diameter of the plate 22 and the number and spacing of theprojections 2li depend on the size of Petri dish i8 intended to be usedand the desired available size of ti e zones 21 of effectiveness. Forexample, seven projections 2@ of a diameter of 6.6 to 10 millimeters maybe arranged in a uniform pattern as shown in FlG. l with their centersspaced apart 3 centimeters from each other on a plate having a diameterof 73 millimeters and intended for use in a Petri dish of 9() or 100millimeters. Smaller or larger numbers may be arranged in regularpatterns on smaller or larger plates as shown in i3 and i4, theprojections on the larger plate of PlG. i4 having a smaller center tocenter spacing. Preferably suitable indicia Si@ such as names (FIG. l)or numbers (PEG. 14) are provided on the plate adjacent the respectiveprojections and are visible from the upper side of the plate to identifythe different agents. The latter are placed on the projections insolutions which dry leaving each agent in a dry state in a layercovering the end 23 of the projection.

The contour of the ends 23 of the projections is especially important inthe use of the plate 22 when it is dropped freely on an inoculatedsurface 17 of agar 16 in a Petri dish i3. Air bubbles thus are removedfrom between the agar and each projection so that the latter seatsfirmly and remains in the same position on the agar and, also, so thatthe agent on the projection diffuses uniformly from the entire area ofthe projection end. After a short time, on the order of two to fiveminutes, the recess 23S forms in the agar beneath each projection andthe film Z5 assumes a uniform thickness. The formation of the recesshelps prevent shifting of the plate on the agar and, in conjunction withthe uniform diffusion of the agent provides controlled test conditionscontributing to accurate results.

With the cover i9 on and the dish 1S inverted as shown in FiG. 6, theprojections 2i? are retained against the agar 16 due to the tensionforces of the film 26 and the lighter weight of the plate. The dishbeing transparent and the plate translucent, the growth of bacteria orother reaction may be easily observed. If it is desircd to remove andreplace the plate in its original position, its proper location isinsured by the orienting lug Z8 and its recess 29.

lt will be apparent that the plate with its projections 20 may be madeinexpensively and thus are disposable after a single use. They are easyto use by personnel with little training and a large number may bestored in a relatively small space. By virtue of the continuous convexcontour of the end 23 of each projection, there results controiledpositioning of the projection on the agar i6 and control of the amountof agent diffused into the agar so as to achieve accurate test results.By utilizing the light weight plate 22 to connect a plurality of theprojections, a corresponding number of agents may be testedsimultaneously while they are maintained at accurate spacing even duringinversion of the Petri dish. The ease and accuracy of the assay thus areenhanced still further.

While the invention is susceptible of various modifications andalternative constructions, the preferred embodiments are shown in thedrawings and are described herein in detail. It is to be understood,however, that such disclosure is not intended to limit the inventionbut, that the aim is to cover all modifications and alternativeconstructions falling within the spirit and scope of the invention asexpressed in the appended claims.

We claim as our invention:

1. In apparatus for assaying the effect of a rst agent on a second agentupon diffusion of the rst agent into an agar-like medium having agenerally ilat exposed surface impregnated with the second agent, asupporting body and an elongated projection extending outwardly fromsaid body and providing an outwardly facing continuous end surface toreceive a layer of said rst agent for diffusion into said medium andreaction with said second agent, said end surface of said projectionbeing formed of non-absorbent material and having a convex contour toengage said medium surface rst at a central portion of the end and thenprogressively to the peripheral portion of the end surface to avoid thetrapping of air between the projection and the medium surface and toinsure uniform distribution of the rst agent throughout the area coveredby the end surface of the projection.

2. In apparatus for use in assaying the effects of a plurality of rstagents of different compositions upon a second agent upon diffusion ofthe first agents into an agar-like medium having a generally at exposedsurface impregnated with the second agent, said apparatus comprising anintegral at disk of thin sheet material having a plurality of spacedportions offset laterally from the plane of the sheet to form hollowprojections extending from one side of the sheet and terminating incontinuous outer end surfaces adapted to receive and support said rstagents in layers on the ends and insure contact of the agents with saidmedium surface over the entire area of each end, said end surfaces beingformed of non-absorbent material and each having a convex contour toengage said medium surface rst at a central portion of the end surfaceand then progressively outwardly to the peripheral portion of the endsurface to avoid the trapping of air between the projection and themedium surface and to insure uniform distribution of the first agentthroughout the area covered by the end surface of the projection.

3. The apparatus of claim 2 in which said disk is formed of alight-weight material such that, when the disk is disposed horizontallybeneath said medium with said projections engaging the medium, theinterfacial tension between the medium and the projections is sucient toovercome the force of gravity and maintain the contact.

4. The apparatus of claim 2 in which in egral projections extendradially and outwardly from the periphery of the disk at angularlyspaced points to engage the upstanding wall of a container for saidmedium and space' the disk inwardly from the wall.

5. The combination of claim 2 in which said hollow projections areconical to permit nesting of the projections of one disk in theprojections of a similar disk with the disks spaced apart.

6. The apparatus of claim 2 in which said end surface is circular andhas a diameter of approximately 6.6 to 10 millimeters and a height of.003 to .006 of an inch.

References Cited bythe Examiner UNITED STATES PATENTS 2,956,931 10/ 1960Goldberg 195-103.5 2,986,497 5/1961 Pagano et al 195-103.5 2,998,3538/1961 Ryan l95-103.5 3,010,880 11/1961 Littman et al. l95-103.5

MORRIS O. WOLK, Primary Examiner.

ANTHONY SCIAMANNA, Examiner'.

1. IN APPARATUS FOR ASSAYING THE EFFECT OF A FIRST AGENT ON A SECONDAGENT UPON DIFFUSION OF THE FIRST AGENT INTO AN AGAR-LIKE MEDIUM HAVINGA GENERALLLY FLAT EXPOSED SURFACE IMPREGNATED WITH THE SECOND AGENT, ASUPPORTING BODY AND AN ELONGATED PROJECTION EXTENDING OUTWARDLY FROMSAID BODY AND PROVIDING AN OUTWARDLY FACING CONTINUOUS END SURFACE TORECEIVE A LAYER OF SAID FIRST AGENT FOR DIFFUSION INTO SAID MEDIUM ANDREACTION WITH SAID SECOND AGENT, SAID END SURFACE OF SAID PROJECTIONBEING FORMED OF NON-ABSORBENT MATERIAL AND HAVING A CONVEXCONTOUR TOENGAGE SAID MEDIUM SURFACE FIRST AT A CENTRAL PORTION OF THE END ANDTHEN PROGRESSIVELY TO THE PERIPHERAL PORTION OF THE END SURFACE TO AVOIDTHE TRAPPING OF AIR BETWEEN THE PROJECTION AND THE MEDIUM SURFACE AND TOINSURE UNIFORM DISTRIBUTION OF THE FIRST AGENT THROUGHOUT THE AREACOVERED BY THE END SURFACE OF THE PROJECTION.